Hydrogen peroxide (H2O2), carbamide peroxide (CP) and peroxo-adducts of inorganic anions such as peroxoborate, peroxodisuiphate and peroxocarbonate as agents for the bleaching of discolored teeth in professional and commercially-available tooth-whitening products such as gels, toothpastes and oral rinses has created much interest regarding their mechanisms of action and redox activity in oral environment as well as adverse effects and safety consideration.
This invention is directed to a novel method of using a therapeutic composition comprising a compound of an α-ketoalkanoic acid salt (pyruvate) and/or its derivatives for the treatment of hydrogen peroxide (H2O2) mediated/induced local inflammation on the tooth structure, surrounding supporting ligaments and oral soft tissue (gingiva and mucosa), during and after external tooth whitening procedure.
The compound is a α-ketoalkanoic acid salt, a physiologically acceptable salt of a α-ketoalkanoic acid, an ester of a α-ketoalkanoic acid salt, or an amide of a α-ketoalkanoic salt. Salts of a α-ketoalkanoic acid, not the free acid, are preferred.
Current Status and Problems of Dental Whitening Procedures Based On H2O2 or Related Oxy Radicals
Exogenous Hydrogen Peroxide is known to cause irritation to many different tissues such as the skin, eyes, esophagus and stomach. Inadvertent exposure of oral soft tissues to a concentrated solution of H2O2 is known to cause extensive tissue damage in the form of vesicle formation and ulceration.
Dental whitening agents such as Hydrogen Peroxide (H2O2) and related oxiradicals such as Carbamide Peroxide (CP) and various other Peroxo-Adducts of inorganic anions are widely used for the bleaching of discolored teeth, both in a professional dental office setting as well as for personal use in additives to oral gels and oral rinsing solutions.
Many studies have examined the mechanisms of action of these and related products, focusing on potential oxidative stress and redox activity in the oral environment as well as possible adverse effects and safety concerns.
One in vitro study showed that H2O2 could penetrate dental enamel, dentine and reach even the pulp chamber.
Furthermore, it has been shown that H2O2, by creating an acidic environment, alters and removes the protective “smear” layer of the dentin as one of the contributing factors to compromise cellular functions.
There is accumulating evidences supporting a direct link between mitochondria which play a key role in energy yielding metabolism and also cell death, the latter as a result of oxidative stress by ROS including H2O2.
Also mucosal tissue safety concerns relate to the potential cytotoxic effects of free radicals produced by the peroxides used in bleaching products: interactions with proteins, lipids and RNA and DNA can lead to cellular damage resulting in apoptosis or direct cytolysis.
Commonly voiced complaints by patients when treated with bleaching agents at commercially available concentrations are considerable tooth sensitivity, discomfort or even direct pain.
Using Carbamide Peroxide, up to 65% of patients report discomfort; when a thermal procedure is added to enhance the bleaching effect, the percentage of patients complaining of pain increases to 91%.
There are isolated reports of patients who have developed oral ulcerations after using 3 percent hydrogen peroxide for 1-2 minutes, 3-5 times daily, while at lower concentrations, changes are less marked or inconspicuous even with continuous exposure.
No effective treatment is yet known or in development to mitigate or perhaps abolish the discomfort and pain of current teeth whitening techniques and procedures.
The most often method to alleviate post-tooth-whitening sensitivity, offered in every professional tooth whitening kit, is the use of fluoride in the affected local area.
Although fluoridated applications are generally effective, the relief of discomfort and sensitivity is not immediate, as it will take weeks to become effective; this delayed effect is probably due to the slow apposition of the fluoridated compounds in the openings of the dentinal tubules.
Bleaching Gels create a high osmotic gradient, given their osmolarity is 17 to 190 times higher than the osmolarity of the dentinal tubular fluid. This creates an osmotic gradient for and out flux of the dentinal tubular fluid resulting in pain (tooth sensitivity) felt by the patient.
As peroxide decomposes, in addition to the formation of molecular oxygen, oxygen ions and radicals (O2−) protons are released. This process can change initially neutral gel to an acidic gel with a pH as low as 3.
The more acidic and the more anhydrous the bleaching gel becomes, the stronger the osmotic gradient pull, and the more forceful is the outflow within the tubule.
It is this outflow of dentinal fluid that is generally assumed to cause an acute discomfort (tooth sensitivity) for the patient.
The relative sensitivities of pulpal, gingival and periodontal ligament cells to toxic effects of hydrogen peroxide are important given the potential for diffusion of hydrogen peroxide through dentine, and thus resulting in pain.
Indeed recent studies have demonstrated that dental bleaching depends on the penetration of hydrogen peroxide (H2O2)-derived free radicals through enamel and into dentin, fragmenting dentin's chromogenic molecules into smaller components.
Previous observations have confirmed that interactions between teeth dentine and bleaching agents involve diffusion and reaction of H2O2 moieties with chromogens (120), so that a direct correlation between the presence of oxidative agents and the penetration potential of H2O2 has already been demonstrated
Considering the fact that enamel permeability for H2O2 is limited and the organic content is about 2%, the most susceptible area of the tooth for H2O2 interaction with organic compounds is at the DentinoEnamel Junction, where the strong oxidizing ability of H2O2 is responsible for the loss of structural organic components.
The reaction between H2O2 and dentin's inorganic compounds could result in the formation of harmful acid by-products such as hydrogen phosphate
These H2O2-induced changes in the organic and inorganic matrix of dentin are considered to be responsible for the inflammatory changes and possible long-term pulpal damage.
Furthermore the role of ROS in the etiology of H2O2-induced tooth sensitivity should be appreciated not be ignored. It has been concluded that, as a result of high concentration hydrogen peroxide employed in external tooth bleaching, an increase of ROS and increase in the activity of the proteolytic enzyme (lysosomal, catepsin B) occurs inside the pulp chamber.
Non-Dental Applications of Alpha-Keto Carboxylates, Particularly Pyruvate, as Oxygen Radical Scavenger and Antioxidant
Pyruvate is a glycolytic metabolite with unique antioxidant, NADH-redox, and energetic features in mammalian cellular systems.
It is a natural α-ketocarboxylate that is also an effective scavenger of reactive oxygen species (oxygen radical scavenger, ROS) with virtually no or little cytotoxicity when applied exogenously in concentrations up to about 10 mM (one hundred fold its physiological level) in short term studies.
Pyruvate-treated human and bovine endothelial cells subjected to a 30 min pulse of oxidative stress by 0.5 mM hydrogen peroxide (H2O2) showed that pyruvate dose-dependently enhanced cellular viability and survival, increasing the bc1-2/bax ratio and stabilizing the cellular glutathione pool.
In isolated liver mitochondria subjected to oxidative stress by simulated ischemia/reperfusion 1 mM pyruvate stabilized respiration, prevented the dissociation of cytochrome C, and enhanced expression of antiapoptotic bc1-2.
In the H2O2-treated endothelial cells pyruvate also transiently increased phosphorylation of ERK1/2 (anti-apoptosis) and prevented the accumulation of phosphorylated p38 mitogen activated kinase (pro-apoptosis).
In L-lactate which generates cytosolic free NADH relative to pyruvate treatment.
These observations point to a redox-related mechanism of action of anti-apoptotic pyruvate.
Enhanced mitochondrial respiration coupled with enhanced bc1-2- and cytochrome C-retention in the isolated liver mitochondria treated with pyruvate indicated improved mitochondrial (inner) membrane stability, enabling improved energetic function and mitochondrial membrane potential Δϕ.
Thus, the cytoprotective mechanism of antioxidant pyruvate is multifactorial, involving cytosolic and mitochondrial redox systems, enhanced survival signaling, and improved mitochondrial inner membrane function.
Pyruvate is also well-documented to strengthen the first line of cellular defense against oxidative stress by increasing the cytosolic NADPH/NADP+ ratio along with an increase in the GSH/GSSG ratio.
In whole organ systems and animal preparations pyruvate has been shown to inhibit myocardial stunning, reduce myocardial infarct size, prolong cortical function during prolonged ischemia, attenuate LPS-induced sepsis.
Pyruvate has also displayed features of metabolic positive inotropy, as in 1-10 millimolar concentrations it not only increases mitochondrial stability and mitochondrial Δϕ but also improves the calcium transients of isolated cardiomyocytes consistent with improved cardiac contractility without apparent cytotoxicity.
Pyruvate as an Endogenous Anti-Inflammatory Agent (Except for Subheadings and References, Passages are Transcribed From R. Bünger Et Al.
A) Physiological and Pathological Generation of Oxiradicals:
ROS such as superoxide anions, hydrogen peroxide, and peroxynitrite are naturally formed in conditions of high cytosolic (and possibly mitochondrial) NADH redox potentials (high free [NADH]/[NAD+] ratio) in the presence of molecular oxygen and, nitric oxide (NO).
It has been estimated that normally up to 2% of mitochondrial oxygen uptake results in formation of ROS (superoxide radical and hydrogen peroxide) via complex I and/or complex III of the mitochondrial respiration chain.
Increased cellular levels of ROS have been implicated in tissue injury due to, e.g., ischemia/reperfusion or hemorrhage/resuscitation, possibly mediated in part via NAD(P)H-dependent membrane-bound NAD(P)H oxidases that generate the superoxide radical.
ROS have also been, found, to alter signaling pathways by oxidizing reactive cysteine residues in specific proteins, reversibly inactivating tyrosine phosphatases and other proteins.
Such protein thiols and sulfhydryl groups after being mildly oxidized by ROS may be re-reduced by reduced glutathione (GSH), one of the most abundant natural cellular antioxidants, whereas continued high levels of oxidative stress probably cause irreversible oxidation of the cysteine thiols and loss of biological activity of peptides and proteins.
B) Pyruvate's Central Role in Intermediary Energy and Redox Metabolism:
Pyruvate is a natural three-carbon glycolytic intermediate with a number of special attributes that are effectively interacting with the redox and energy metabolism of the cell.
Besides its central role in amino acid metabolism, it is substrate and allosteric activator of the mitochondrial pyruvate dehydrogenase and of the CO2-fixing mitochondrial carboxylase, which renders pyruvate the anaplerotic precursor of mitochondrial oxaloacetate and citrate providing substrate for the NADPH-dependent isocitrate dehydrogenase (ICDH) and maintaining the level of citric acid cycle intermediates.
With respect to the redox systems in the cytosol, pyruvate is the precursor of L-lactate via the abundant near-equilibrium lactate dehydrogenase, a reaction that produces cytosolic free NAD+ thus effectively competing for NADH substrate for superoxide generating NAD(P)H oxidases.
This only recently recognized feature identifies pyruvate as a natural antioxidant that additionally stabilizes the cellular ATP pool and can raise the cytosolic phosphorylation potential substantially.
C) Pyruvate as Oxiradical Scavenger:
Pyruvate is also a very effective non-enzymatic scavenger of intra- and extracellular oxyradicals such as hydrogen peroxide and particularly of peroxynitrite (rate constant 88-100 mol−1*s−1 at pH 7.4, 37° C.,), an especially cytotoxic oxyradical.
In the reaction between pyruvate and peroxynitrite pyruvate decarboxylates to acetate at a speed that is about one order of magnitude faster than that with H2O2; peroxynitrite itself can be formed extremely fast from NO and the superoxide anion with a rate constant of 4-7*109/mol*sec.
D) Pyruvate as Natural Cytoprotective Antioxidant Molecule:
Antioxidant pyruvate has been found strikingly effective and beneficial in numerous experimental and some short-term clinical pilot studies, with little or no detectable toxicity.
For example pyruvate is cytoprotective in ischemia/reperfusion or infarct/stunning paradigms in tissues such as heart, brain, kidney, liver, lens, vascular endothelium, and intestine.
The first published pilot study in heart failure patients with dilated cardiomyopathy also demonstrated beneficial efficacy of intracoronary pyruvate with no obvious toxicity.
Similarly, pyruvate cardioplegia reduced mortality and morbidity in a recent study on elective coronary revascularization in cardiac patients as well as in in-vitro perfused hearts.
E) Mechanisms of Pyruvate Cytoprotection:
The pyruvate protective mechanisms are likely multifactorial, mediated by improved cellular NADH- and thiol redox status alone or combined with enhanced mitochondrial anaplerosis and energetics. Pyruvate treatment inhibits the basal and the reperfusion burst of ROS, decreases cytosolic free NADH levels and thus NAD(P)H oxidase activity, preserves or increases the cytosolic NADPH and GSH pools, stabilizes cellular ATP and hence intracellular pH, raises the thermodynamic ATP phosphorylation potential, and prevents the mitochondrial permeability transition in reperfusion following cardiac ischemia.
There is another less-well recognized antioxidant mechanism of pyruvate which is mediated by the anaplerotic mitochondrial pyruvate carboxylase; in this pathway pyruvate stimulates formation of matrix oxaloacetate and citrate; the latter, after export into the cytosol, can exert two major effects with respect to the cellular antioxidant status: i) allosteric inhibition of the phosphofructokinase causing upstream accumulation of glucose-6-phosphate and thus enhanced potential for oxidative pentose phosphate pathway (PPP) flux required for reductive syntheses and reduction of cytosolic NADP and oxidized glutathione (GSSG) to NADPH and GSH, respectively; ii) direct stimulation of the NADPH-dependent isocitrate dehydrogenase of the cytosol which also yields NADPH available for reduction of GSSG.